The cells making up the various tissues and organs systems are held together by specific molecules that essentially serve as “biological glue” conferring shape, structure, rigidity or plasticity. During embryogenesis, these biological molecules or extracellular matrix (ECM) proteins serve as “tracks” and direct cells to the appropriate vicinity within the embryo to give rise to tissues and organ systems. ECM proteins also play a prominent role during wound healing, and are involved in directing other cellular processes such as proliferation, survival, and differentiation. Failure of cells to interact with the appropriate biological surface or molecule can be detrimental to the faith of the cells and can contribute to cancer cell metastases.
There are several methods for assessing and quantifying cellular adhesion and spreading on an ECM coated surface. The most widely used method is to apply the cells onto surfaces coated with appropriate ECM components, allow the cells to attach and adhere for a specified length of time and wash the unbound cells. The attached cells are then fixed, labeled with fluorescent reagent such as rhodamine phalloidin and visualized using an epi-fluorescent microscope or an epi-fluorescent confocal microscope. Alternatively, the cells can be labeled with a dye such as crystal violet and quantified by either counting the stained cells using a light microscope or solubilizing the stain and obtaining absorbance reading using a spectrophotometer. Cells can also be pre-labeled with a fluorescent dye for live cells such as 6-carboxyfluorescein diacetate (CFDA) and then applied to appropriate ECM-coated surface. The unbound cells are washed off and the bound cells are quantified using a plate reader. An additional method for assessing the role of integrins and other adhesion proteins is to coat different surfaces with antibodies or peptides which are specific for the various receptors and then seed the cells which are expressing the appropriate integrin receptors. The interaction of integrin receptor on the cell surface with the antibody or peptide-coated surface will allow the cells to adhere and undergo specific morphological and biological changes which can then be assessed by using cell biological techniques discussed above.
While the assays just described for assessing and quantifying cell adhesion have been informative, there are certain caveats associated with each of these assays. For example, each of the assays described are end-point assays which provide a “snapshot” of the adhesion process. All the assays involve pre-labeling or post-labeling of the cells and also involve fixation and permeabilization leading to destruction of the cell. In this application we describe a label-free real-time assay using electronic cell sensor technology (RT-CES system) which addresses some of the major limitations of the current in vitro assays for assessing the interaction of bio-molecular coated surfaces with target cells. Furthermore, because the readout is non-invasive it precludes the need for fixation and lysis of the cells and allows for acquisition of information for biological events occurring after adhesion and spreading, such as proliferation and differentiation.